Induced pluripotent stem cell derived pericytes respond to mediators of proliferation and contractility

Background Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood–brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). Methods We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. Results iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators. Conclusions iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-024-03671-x.


Pericyte morphology analysis
Phase contrast micrographs of neural crest and mesoderm iPericytes were captured with the 4x objec�ve on a Zeiss microscope using a Camera AxioCam ICc5 (Imbros).Each individual pericyte was manually assigned to a morphological subtype based on their appearance according to the criteria outlined previously (1).Percentage of each morphology within cultures was calculated from N = 746 (neural crest iPericytes) and 759 (mesoderm iPericytes) cells analysed across one culture for each differen�a�on method.

Pericyte expression analysis
To es�mate the percentage of pericytes that expressed PDGFRβ and CD13, fluorescent images were captured with the 20x objec�ve on an Olympus FV3000 Super Resolu�on confocal laser scanning microscope (Olympus, Japan) with iden�cal se�ngs for each marker between HBVPs, mesoderm iPericytes, and neural crest iPericytes.Images were processed in ImageJ using despeckle, then B&C manually thresholded for HBVPs un�l cellular autofluorescence was minimal.B&C thresholds were propagated to mesoderm and neural crest iPericyte images, then cells were manually assigned posi�ve expression if cytosolic (CD13) labelling and cytosolic plus puncta (PDGFRβ) labelling were present above threshold in each DAPI posi�ve cell, or nega�ve if labelling was not present.A minimum of 250 DAPI posi�ve cells was counted for each group.

Comparison of three iPSC lines differentiated to three mesoderm iPericyte lines
Following library prepara�on for each sample, RNA was sequenced to a minimum depth of 20 million reads with 150 base paired end lengths.Raw sequencing data was processed with TrimGalore (v0.6.7)(2).The sequencing adapter (CTGTCTCTTATACACATCT) was trimmed from each read and 1 base pair was removed from the 5' end of each read.A read quality Phred score threshold of 30 was used to remove low quality reads.Reads with a length of less than 25 base pairs a�er trimming were discarded.The quality of the sequencing data was then evaluated using FastQC (v.0.11.9) (3), and all data met the necessary quality metrics for analysis.
Gene expression was quan�fied using Salmon (v.1.8.0)(4).The Salmon indexed transcriptome for Homo sapiens (GRCh38) was downloaded using RefGenie (v.0.12.1) (5) and Salmon used to quan�fy gene expression against this transcriptome in mapping-based mode.The sequencing library type was ISR.Within Salmon, --validateMappings was employed to improve the sensi�vity and specificity of the read mapping (and thus quan�fica�on accuracy); --seqBias and --gcBias were employed to enable Salmon to learn and correct for sequencespecific biases and fragment-level GC biases respec�vely.The result of this analysis was per sample quan�fica�on of transcript expression.
Differen�al gene expression analysis was performed using DESeq2 (v1.34.0) (6) to compare gene expression between iPSCs and mesoderm-iPericytes.Tximport (v1.27.1) (7) was used to import transcript-level abundance into R (v.4.3.0) and summarise abundance to the genelevel.Genes with less than 1 read across each group were filtered out.Heatmaps were generated using pheatmap (v.1.0.12) (8).impedance to electron flow, thus increased resistance, which is interpreted as a higher 'Cell Index'.Contracted cells have reduced cell volume meaning less impedance to electron flow resul�ng in reduced resistance, which is interpreted as lower 'Cell Index'.(B) For contrac�le studies, cell index for each condi�on was measured every minute for 2h and was normalised to �me point 0, when vasoac�ve agents were added.To quan�fy differences between groups, three measures were extracted from the normalised cell index over �me: slope (m) shows the rate of contrac�on over the first 10 mins and is calculated as the slope of the linear equa�on y = mx+b for each replicate from t = 0 to t = 10; area under the curve (AUC) shows the cumula�ve change in contrac�on over �me and is calculated as the area (A) under the curve between t = 0 and t = 20 mins; ∆ cell index shows the change in cell index from baseline at specific �me points, e.g. at the �me of maximum contrac�on, contrac�on a�er 2h.

Figure S1 .
Figure S1.iPSCs can be differen�ated into pericytes through the neural crest and mesoderm induc�on pathways.iPSCs were placed in either mesoderm induc�on media or neural crest induc�on media for 5 days.Media was then replaced with complete pericyte media for another 5 days to generate neural crest iPericytes or mesoderm iPericytes.Protocol adapted from Faal et al. (13).

Figure S3 .
Figure S3.iPericytes display heterogenous morphology consistent with HBVP cells in vitro.Pericyte morphology was classified following the criteria outlined in Brown et al. (1).(A) Both neural crest iPericytes and mesoderm iPericytes display the five key morphological classifica�ons of pericytes in vitro: balling, spindle, sheet, circular, and standard.(B) Quan�fica�on of the percentage of each pericyte morphology subtype in neural crest iPericyte culture (746 cells) and mesoderm iPericyte culture (759 cells).Scale = 20 µm.

Figure S6 .
Figure S6.Schema�c of pericyte contrac�on measurement and analysis using the xCelligence system.To test contrac�le func�on of neural crest iPericytes and mesoderm iPericytes in comparison to HBVPs, the contrac�le response of cells was measured using an xCelligence system.(A) Cells were plated in specialised cell culture plates with electrodes which measure electron flow.Relaxed cells have greater cell volume meaning larger